Active and passive transport are critical process for normal cell metabolism, including the maintenance of ion-gradients, osmotic balance, membrane potentials, and apoptosis. In spite of their widespread importance, the molecular mechanisms that lead to transport have not been characterized, which is in part due to the difficulty associated with obtaining structural information on membrane proteins. The work that is described in this proposal is directed at investigating the mechanisms of action of two transport systems. BtuB is an outer membrane transport protein for vitamin B12 found in gram negative bacteria, and it obtains its energy for transport by coupling to the inner membrane protein TonB. Using site-directed spin-labeling and EPR spectroscopy, we will test proposals for the molecular mechanisms of transport in BtuB. This class of membrane proteins is of fundamental interest because it is the only class of membrane active transport proteins for which high-resolution structural models have been obtained; as a result, they are likely to be the first active transport systems for which detailed molecular mechanisms will be obtained. In addition to the accumulation of nutrients, this class of proteins functions in the uptake of bactericidal agents, such as colicins, phages and small molecule antibiotics. The widespread importance of these TonB dependent systems for bacterial function makes them probable targets for the development of new classes of antibiotics. A second system that will be studied is alamethicin, a peptide antibiotic that forms voltage-dependent ion channels in bilayers. Voltage- dependent events in membrane proteins are also of widespread importance, but have not been characterized at a molecular level. Alamethicin belongs to a larger class of membrane active peptides having important antibiotic, fungicidal, hemolytic and tumoricidal activities. In addition to its voltage-dependence, alamethicin has received attention because it appears to be selective towards certain organisms. In the work proposed here we will develop a method to apply electric fields across vesicle and supported bilayers and will use NMR, EPR and FTIR to test mechanisms for the voltage-dependence in this peptide. We will also test mechanisms that could account for the selectivity of this antibiotic against certain membranes.